Vyas Rajan, Reed Andrew J, Raper Austin T, Zahurancik Walter J, Wallenmeyer Petra C, Suo Zucai
Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH 43210, USA.
The Ohio State Biochemistry Program, The Ohio State University, Columbus, OH 43210, USA.
Nucleic Acids Res. 2017 Jun 2;45(10):6228-6237. doi: 10.1093/nar/gkx252.
Nucleoside reverse transcriptase inhibitors (NRTIs) with L-stereochemistry have long been an effective treatment for viral infections because of the strong D-stereoselectivity exhibited by human DNA polymerases relative to viral reverse transcriptases. The D-stereoselectivity of DNA polymerases has only recently been explored structurally and all three DNA polymerases studied to date have demonstrated unique stereochemical selection mechanisms. Here, we have solved structures of human DNA polymerase β (hPolβ), in complex with single-nucleotide gapped DNA and L-nucleotides and performed pre-steady-state kinetic analysis to determine the D-stereoselectivity mechanism of hPolβ. Beyond a similar 180° rotation of the L-nucleotide ribose ring seen in other studies, the pre-catalytic ternary crystal structures of hPolβ, DNA and L-dCTP or the triphosphate forms of antiviral drugs lamivudine ((-)3TC-TP) and emtricitabine ((-)FTC-TP) provide little structural evidence to suggest that hPolβ follows the previously characterized mechanisms of D-stereoselectivity. Instead, hPolβ discriminates against L-stereochemistry through accumulation of several active site rearrangements that lead to a decreased nucleotide binding affinity and incorporation rate. The two NRTIs escape some of the active site selection through the base and sugar modifications but are selected against through the inability of hPolβ to complete thumb domain closure.
由于人类DNA聚合酶相对于病毒逆转录酶表现出强烈的D-立体选择性，具有L-立体化学结构的核苷类逆转录酶抑制剂（NRTIs）长期以来一直是治疗病毒感染的有效药物。DNA聚合酶的D-立体选择性直到最近才从结构上进行探索，迄今为止研究的所有三种DNA聚合酶都展示了独特的立体化学选择机制。在此，我们解析了人类DNA聚合酶β（hPolβ）与单核苷酸缺口DNA和L-核苷酸形成复合物的结构，并进行了预稳态动力学分析，以确定hPolβ的D-立体选择性机制。除了在其他研究中看到的L-核苷酸核糖环类似的180°旋转外，hPolβ、DNA和L-dCTP或抗病毒药物拉米夫定（（-）3TC-TP）和恩曲他滨（（-）FTC-TP）的三磷酸形式的催化前三元晶体结构几乎没有提供结构证据表明hPolβ遵循先前表征的D-立体选择性机制。相反，hPolβ通过积累几种活性位点重排来区分L-立体化学结构，这些重排会导致核苷酸结合亲和力和掺入率降低。这两种NRTIs通过碱基和糖修饰逃避了一些活性位点选择，但由于hPolβ无法完成拇指结构域闭合而被排除。
